Acta Medica Okayama 56巻 4号

The ferric nitrilotriacetate-induced carcinogenesis model is unique in that reactive oxygen species-free radicals are involved in the carcinogenic process. But the effects of iron-withdrawal in the progression of renal cell carcinoma are not well understood. We performed repeated phlebotomies on animals that had been administered ferric nitrilotriacetate in the initiation stage of renal cell carcinoma (phlebotomy group), and compared the development of renal tumors with those not receiving repeated phlebotomies (non-phlebotomy group). Ferric nitrilotriacetate-treated male Wistar rats were randomly divided into 2 groups: a phlebotomy group (21 rats) and a non-phlebotomy group (17 rats). Ten age-adjusted normal rats were also observed as a normal group. Hematocrit was maintained under 25% in the phlebotomy group. Hematocrit levels in the normal group and in the non-phlebotomy group were not significantly different. As a result, the incidence of renal cell carcinoma was not significantly different between phlebotomy and non-phlebotomy animals. However, the total weight of the renal cell carcinoma was significantly heavier in the animals from non-phlebotomy group than in those from the phlebotomy group (23.64 g +/- 18.54 vs. 54.40 g +/- 42.40, P < 0.05). The present study demonstrated that phlebotomy after the administration of ferric nitrilotriacetate did not reduce the incidence of renal cell carcinoma. In addition, we showed that iron withdrawal at the promotion stage of carcinogenesis will retard tumor growth.

We have previously developed an enzyme-linked immunosorbent assay (ELISA) to measure stool decay-accelerating factor (DAF) and found that stool DAF concentrations were significantly elevated in patients with colorectal cancer, suggesting that the measurement of stool DAF may be valuable for the detection of colorectal cancer. In order to refine the assay for the measurement of stool DAF, we investigated 1) effects of centrifugation of stool samples, 2) effects of detergents, and 3) adequate combination of various anti-DAF monoclonal antibodies for the ELISA system using only monoclonal antibodies. We found that high-speed centrifugation could be omitted and that only the removal of large undigested food residues by centrifugation of short duration in a low-speed benchtop microcentrifuge sufficed to adequately prepare the stool samples. Addition of 2 detergents, octyl beta-glucoside and sodium deoxycholate, known to solubilize glycosyl-phosphatidylinositol-anchored proteins such as DAF, did not influence stool DAF values. By using 2 mouse anti-DAF monoclonal antibodies (clone 4F11 and 1C6), we were able to achieve a stable ELISA for the measurement of stool DAF using a uniform source of antibodies. The results should allow us to consistently apply the DAF assay for routine use in the detection of colorectal cancer.

The preS2 region of the hepatitis B virus (HBV) has been reported to have human polymerized albumin receptor (PAR) activity, which correlates with viral replication. Here, we studied the genomic sequence of the preS region from rare patients lacking PAR activity, despite active viral replication. PAR and DNA polymerase activity was identified in 178 HBe antigen-positive HBV carriers, and a significant correlation between 2 markers was shown, except in 2 hepatitis patients lacking PAR activity. Nucleotide sequences of the preS region of HBV from both patients were examined by direct sequencing of PCR products. In one patient, a 45-base deletion was found to overlap half of the putative polymerized human albumin binding site in the preS2 region. In the other patient, a point mutation at the first nucleotide of the start codon of the preS2 region of HBV was found. There was no such genomic change in the 3 control HBV sequences. These results indicate that the preS2 region is necessary for binding of polymerized human albumin, and this is the first report of naturally existing mutant virus with no or low PAR activity.

We developed a monoclonal antibody (MoAb) (clone 5E8) against an antigen on the bile canalicular membrane of rat hepatocyte. By immunoblotting, MoAb 5E8 detected a band of 110 kD. In this study, we used the phage display technique to identify the target antigen recognized by MoAb 5E8. We screened a random phage display library expressing 12-mer peptide sequences and identified a peptide sequence, FHFNPYTGHPLT, as an epitope. We compared this peptide sequence with those of dipeptidyl peptidase IV (DPP IV, E.C.3.4.14.5) and Cell-CAM105, which proteins were located by a database search based on the information of tissue localization and approximate molecular weight of the MoAb 5E8 antigen, and sequence similarity with a region in DPP IV (amino acids 225-233) but not with Cell-CAM105 was found. In addition, we immunohistochemically stained various tissues (liver, small intestine, and kidney) of Japanese Fischer 344 rats, known to be deficient for DPP IV, with MoAb 5E8 and showed that the expression of MoAb 5E8 antigen was negligible or weak. In contrast, tissues sampled from the same organs of Sprague-Dawley rats, known to express DPP IV, were positively stained. These findings suggest that the antigen recognized by MoAb 5E8 is DDPIV and its major epitope is located in amino acids at positions 225-233.

To compare morphological changes in the umbilical cords from chronic hypertensive and preeclamptic patients having normal or pathological umbilical artery Doppler ultrasonographic results. Umbilical cords from 34 normotensive, 31 chronic hypertensive and 70 preeclamptic women with normal and abnormal Doppler flow velocity waveforms (FVW) at 35-40 gestational weeks were studied. Morphological changes in the umbilical cords were examined on formalin-fixed, paraffin-embedded sections. The total umbilical cord area, total vessel area, and wall thickness of umbilical vessels were measured in systematic random samples using unbiased stereology methods. An ANOVA test was used for statistical analysis. In the chronic hypertensive and preeclamptic groups with normal Doppler FVW, the thickness of the umbilical cord vessels remained nearly constant, whereas both the total area and the lumen area were reduced. These changes correlate with the histopathological findings, suggesting a mainly vasoconstrictive effect. By contrast, analysis of the preeclamptic group with pathologic Doppler FVW showed a comparable reduction of all parameters of the umbilical cord. Histopathological findings were related to smaller, contracted smooth muscle cells of the vessel wall, which is suggestive of a predominant hypoplastic mechanism. As a result of reduced uteroplacental perfusion, fetal hypoxia and intrauterine growth retardation become unavoidable in preeclampsia. The histopathological changes in the umbilical cord between the chronic hypertensive and preeclamptic patients depend on the Doppler results. In conclusion, the umbilical artery Doppler FVW indices provide good values for predicting intrauterine growth retardation in preeclamptic patients.

Dynamin is a protein essential to endocytosis. Dynamin 2, a dynamin isoform, is expressed most intensely in testicular tissue; however, precise localization has never been studied. Therefore, we investigated the expression of dynamin 2 in rat testicular tissue using immunohistochemical methods, and discuss here the physiological function of this protein. Testicular tissues were obtained from Wistar rats at 10, 21 and 63 days of age. Immunohistochemistrical examination and Western blot analysis were conducted using dynamin 2 specific antibody. Western blot analysis showed that expression in 21- and 63-day-old rats was more intense than that in 10-day-old rats. Dynamin 2 expression was observed using immunohistochemical method in the seminiferous tubules of all rats. In the 63-day-old rats, the expression was intense, especially in spermatids in the earlier maturation stages and in spermatocytes, and was observed in Sertoli cells. However, in spermatids, the expression gradually declined as spermatids matured to spermatozoa. In the 21-day-old rats, the expression was evident in spermatocytes and Sertoli cells, but that in the 10-day-old rats was weak. Intense expression of dynamin 2 during spermatogenesis suggests that this protein plays an important role in this process.